exRNA Communication Mechanisms, Biomarkers, and Therapies

Extracellular RNA Communication: 
Mechanisms, Biomarkers,  
and Therapies
Friday 20 October, 2017
Scientiﬁc Outreach Coordinator
Extracellular RNA Communication Consortium
Roger P. Alexander
Senior Staff Scientist
Paciﬁc Northwest Diabetes Research Institute

OVERVIEW exRNA PROFILES BIOGENESIS BIOMARKERS THERAPIES RESOURCES
The Extracellular RNA Communication Consortium
2
OVERVIEW
•  RNA was once thought to exist in a stable form only
inside cells, serving as an intermediate in the translation
from genes to proteins.
•  However, recent research has indicated that RNAs can
play a role in a variety of complex cellular functions,
including newly discovered mechanisms of cell-to-cell
communication.
•  RNA can be exported from cells in extracellular vesicles
(EVs) or bound to lipids or proteins, circulating through
the body and affecting distant cells and tissues. These
extracellular RNAs (exRNAs) may also be absorbed from
food, from the microbiome, or from the environment.

The Extracellular RNA Commuication Consortium
3
•  The NIH Common Fund identiﬁed this new paradigm of intercellular
and inter-species information exchange as an important area of
inquiry, so in 2013 it launched the Extracellular RNA Communication
Consortium (ERCC).
•  The goals of the ERCC are
–  to discover fundamental biological principles about the
mechanisms of exRNA generation, secretion, and transport
–  to investigate the potential for using exRNAs in the clinic as
•  therapeutic molecules or
•  biomarkers of disease
–  to identify and develop a catalog of exRNAs found in normal
human body ﬂuids
OVERVIEW

Outline
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1.  exRNA proﬁles in different bioﬂuids
Examples of consortium work on
2.  Biogenesis:
KRAS-dependent sorting of miRNA into exosomes
3.  Biomarkers for glioblastoma
4.  Therapy: Stem cell EVs rescue mice from liver failure
5.  Consortium resources
–  exRNA Portal
–  exRNA Atlas
–  Virtual Biorepository
OVERVIEW exRNA PROFILES BIOGENESIS BIOMARKERS THERAPIES RESOURCESOVERVIEW

Outline
5
1.  exRNA proﬁles in different bioﬂuids
Examples of consortium work on
2.  Biogenesis:
KRAS-dependent sorting of miRNA into exosomes
3.  Biomarkers for glioblastoma
4.  Therapy: Stem cell EVs rescue mice from liver failure
5.  Consortium resources
–  exRNA Portal
–  exRNA Atlas
–  Virtual Biorepository
OVERVIEW exRNA PROFILES BIOGENESIS BIOMARKERS THERAPIES RESOURCESOVERVIEW
(Y-RNA is involved!)

Yeri, A. et al. Total extracellular small RNA profiles from plasma, saliva, and urine of healthy subjects. Sci. Rep. (2017) 7: 44061.
RNA Profiles in different biofluids
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exRNA PROFILES
Plasma, urine, and saliva data are
from Yeri et al. 2017
CSF data is from the Jensen lab,
unpublished.
50-170 samples
of small RNA
purified from
whole biofluid

7
exRNA PROFILES
• On average, CSF and plasma have
much higher fractions of miRNA than
urine and saliva, about 25%.
• To date, most biomarker research
has focused on identifying miRNA
biomarkers of disease because of
their well-known function in negative
regulation of gene expression.

8
exRNA PROFILES
• Over 60% of the mappable RNA in
plasma is YRNA, almost entirely from
the 5’ end of RNY4. The human
genome contains 4 YRNA genes and
52 YRNA pseudogenes. YRNAs play
a role in chromatin interaction during
DNA replication, but their role as
exRNAs is not yet known.

9
exRNA PROFILES
• Over half of the RNAs in urine map
to multiple loci, mainly overlap piRNA
and tRNA fragments (tRFs).
• Otherwise, exRNA in urine consists
mainly of tRNA fragments.
• CSF also has a high fraction of
tRNA fragments.

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exRNA PROFILES
• Saliva in particular includes a very
large fraction of exogenous RNA
from the oral microbiome (> 90%).

Kaczor-Urbanowicz, K.E.. et al.
Novel approaches for bioinformatic analysis of
salivary RNA sequencing data for development.
Bioinformatics (2017)
doi: 10.1093/bioinformatics/btx504
Exogenous RNA in saliva
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exRNA PROFILES
Saliva

Exogenous RNA in saliva
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exRNA PROFILES
Saliva
Symposium at AADR 2018
(annual meeting of the
American Association
for Dental Research)
Salivaomics: Saliva Extracellular RNA (exRNA)
& the Saliva Proteome Wiki

Biogenesis and delivery of exRNA
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BIOGENESIS
Biogenesis, delivery, and function of extracellular RNA. Patton, J.G. et al.
J. Extracellular Vesicles (2015) 4:27494. doi: 10.3402/jev.v4.27494.

KRAS-dependent sorting of miRNA into EVs
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BIOGENESIS
KRAS-MEK Signaling Controls Ago2 Sorting into Exosomes. McKenzie, A.J. et al.
Cell Reports (2016) 15: 978–987.
When KRAS is hyper-active (MutDKO-1 cells), it inhibits Argonaute2
(Ago2) localization to multivesicular endosomes (MVEs).
MVE
marker
P body
marker
WT, low
KRAS
activity
mutant
hyper-
active
KRAS

KRAS-dependent sorting of miRNA into EVs
15
BIOGENESIS
KRAS-MEK Signaling Controls Ago2 Sorting into Exosomes. McKenzie, A.J. et al.
Cell Reports (2016) 15: 978–987.
When KRAS is hyper-active,
it initiates a MAP kinase cascade
that phosphorylates Ago-2 on S387
which inhibits Ago2 localization to
MVEs
and decreases secretion
in exosomes of Ago2
and miRNAs bound to it.

Proving delivery of ncRNA to target cells
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DELIVERY
exRNA.org/About
For the complete story, watch James Patton’s seminar at
Hyper-active KRAS
in colorectal cancer cells
shunts miR-100 into EVs,
which transform target cells
when ingested.

Glioblastoma EVs shape the tumor micro-environment
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BIOMARKERS
Skog, J. et al. Glioblastoma microvesicles transport RNA and proteins that promote tumour growth
and provide diagnostic biomarkers. Nat. Cell Biol. (2008) 10: 1470-1476.
•  Glioblastomas release copious numbers of
extracellular vesicles (EVs).

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•  Those EVs are taken up by surrounding myeloid cells— microglia and macrophages.
•  Normally functioning microglia are nurturers of self
but sentinels and warriors against other.
•  Tumor EV uptake by microglia turns off their sentinel and warrior pathways and
activates nurturing of tumor cells.
BIOMARKERS
Glioblastoma EVs shape the tumor micro-environment
Skog, J. et al. Glioblastoma microvesicles transport RNA and proteins that promote tumour growth
and provide diagnostic biomarkers. Nat. Cell Biol. (2008) 10: 1470-1476.
Glioblastoma cells grown for 3 days same, but supplemented with
glioblastoma microvesicles

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BIOMARKERS
•  RNA and EVs from glioblastoma
show up in CSF
•  Can CSF miRNA be used as
biomarkers for glioblastoma?
•  ERCC researchers Fred Hochberg,
Ying Mao, Bob Carter, and Clark Chen
analyzed miRNA from 135 CSF
samples in 3 cohorts
using TaqMan OpenArray®
Human MicroRNA Panels
exRNA biomarkers for glioblastoma
Akers, J.C. et al. A cerebrospinal ﬂuid microRNA signature as biomarker for glioblastoma. Oncotarget (2017) 8: 68769-68779.

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BIOMARKERS
•  miRNAs with levels that differed between
GB and healthy CSF were identiﬁed using
the criteria
FDR < 0.2 and log(fold-change) > 2
5 enriched in GB CSF
miR-21
miR-218
miR-193b
miR-331
miR-374a
4 depleted in GB CSF
miR-548c
miR-520f
miR-27b
miR-130b
•  24 miRNAs were selected
•  LASSO regression down-selected to 9 miRNA:

21
BIOMARKERS
Performance of 9 miRNA classiﬁer
(validated in 60 CSF samples from 2 cohorts)

22
BIOMARKERS
•  miRNA profiles in lumbar and cisternal
CSF -- fluid collected from the spine or
the base of the neck, respectively --
are significantly different, which is
problematic, since cisternal CSF is
much more difficult to collect.
•  On the other hand, they also found that RNAs
extracted from raw CSF had a similar profile and
diagnostic power as RNAs extracted from
vesicles after an initial EV purification step.
Figueroa, J.M. et al. Detection of wtEGFR Amplification and EGFRvIII Mutation in CSF-Derived Extracellular Vesicles of
Glioblastoma Patients. Neuro. Oncol. (2017) Advance online publication. doi: 10.1093/neuonc/nox085.

Mice with lethal liver failure rescued  
by extracellular vesicles from stem cells
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•  Co-administration of Tumor Necrosis Factor alpha (TNF-α)
and D-galactosamine (Dgal) results in liver failure,
characterized by
–  tremulousness
–  impaired ability to walk straight and rise from lying down
–  loss of eyelash reﬂex
–  eventual coma and death
•  Liver failure can be rescued by administration of bone-
marrow-derived mesenchymal stem cells (MSCs).
•  What about EVs from MSCs?
THERAPIES
Extracellular vesicles from bone marrow-derived mesenchymal stem cells improve survival from
lethal hepatic failure in mice. Haga H, Yan IK, Takahashi K, Matsuda A, Patel T.
Stem Cells Transl. Med. (2017) 6:1262-1272. doi: 10.1002/sctm.16-0226.

24
•  x
THERAPIES
In mice with TNFα/Dgal-
induced liver failure,
mMSC-EVs are taken up
speciﬁcally by liver and spleen,
and not other organs.
IP
control
IP
liver injury
IV
control
IV
liver injury

25
THERAPIES
n=8
n=6
n=6
n=8
n=7
n=8
n=8
n=7
n=7
n=6
n=4
n=4
n=4
57% survival at 24 hrs
37.5% survival at 24 hrs

26
THERAPIES
lncRNA expression was
assessed by comparing RT-qPCR
in hMSC and hMSC-EV
...Turning an EV story into an exRNA story...
YRNA-1

27
THERAPIES
Extracellular vesicles from bone marrow-derived mesenchymal stem cells improve
survival from lethal hepatic failure in mice.
Haga H, Yan IK, Takahashi K, Matsuda A, Patel T.
Caspase-3 (brown) is a marker of apoptosis.
Scale bar = 200 µM
YRNA-1

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THERAPIES
Scale bar = 200 µM
•  Actinomycin-D induces apoptosis
Caspase 3/7 assay
of human hepatocytes
YRNA-1

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THERAPIES
Scale bar = 200 µM
•  hMSC-EVs reduce apoptosis
Caspase 3/7 assay
YRNA-1

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THERAPIES
Scale bar = 200 µM
•  hMSC-EVs reduce apoptosis
•  Loss of YRNA1 via siRNA knockdown
partially blocks the effectiveness of
hMSC-EVs
Caspase 3/7 assay
YRNA-1

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THERAPIES

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OVERVIEW exRNA PROFILES BIOGENESIS BIOMARKERS THERAPIES RESOURCESRESOURCES
exRNA.org
exRNA-Atlas.org
small RNA-seq analysis pipeline
1)
2)
3)
4) (See poster #59.)

33

exceRpt small RNA-seq
analysis pipeline
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Upcoming Conferences
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Newry, Maine August 19 - 24, 2018
Keystone Symposium on Exosomes/Microvesicles:
Heterogeneity, Biogenesis, Function, and Therapeutic
Developments Ÿ Breckenridge, Colorado Ÿ June 4-8, 2018
RNA Nanotechnology
Ventura, California Ÿ January 2019

exRNA Communication Mechanisms, Biomarkers, and Therapies

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